· was used in that stage of the research. ... exhibits both permanent and temporary shallow...

1 23

ExtremophilesMicrobial Life Under ExtremeConditions ISSN 1431-0651 ExtremophilesDOI 10.1007/s00792-012-0452-1

Enrichment of arsenic transforming andresistant heterotrophic bacteria fromsediments of two salt lakes in NorthernChile

José Lara, Lorena Escudero González,Marcela Ferrero, Guillermo ChongDíaz, Carlos Pedrós-Alió & CeciliaDemergasso

1 23

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ORIGINAL PAPER

Enrichment of arsenic transforming and resistant heterotrophicbacteria from sediments of two salt lakes in Northern Chile

Jose Lara • Lorena Escudero Gonzalez •

Marcela Ferrero • Guillermo Chong Dıaz •

Carlos Pedros-Alio • Cecilia Demergasso

Received: 13 January 2012 / Accepted: 2 April 2012

� Springer 2012

Abstract Microbial populations are involved in the arsenic

biogeochemical cycle in catalyzing arsenic transformations

and playing indirect roles. To investigate which ecotypes

among the diverse microbial communities could have a role

in cycling arsenic in salt lakes in Northern Chile and to obtain

clues to facilitate their isolation in pure culture, sediment

samples from Salar de Ascotan and Salar de Atacama were

cultured in diluted LB medium amended with NaCl and

arsenic, at different incubation conditions. The samples and

the cultures were analyzed by nucleic acid extraction,

fingerprinting analysis, and sequencing. Microbial reduction

of As was evidenced in all the enrichments carried out in

anaerobiosis. The results revealed that the incubation fac-

tors were more important for determining the microbial

community structure than arsenic species and concentra-

tions. The predominant microorganisms in enrichments

from both sediments belonged to the Firmicutes and Pro-

teobacteria phyla, but most of the bacterial ecotypes were

confined to only one system. The occurrence of an active

arsenic biogeochemical cycle was suggested in the system

with the highest arsenic content that included populations

compatible with microorganisms able to transform arsenic

for energy conservation, accumulate arsenic, produce H2,

H2S and acetic acid (potential sources of electrons for

arsenic reduction) and tolerate high arsenic levels.

Keywords Arsenic microbial metabolisms �Arsenic-resistant microorganisms � Northern Chile

Introduction

Arsenic is widely present in water, rocks, and soils in many

different areas of the world, including the Antofagasta

region, Northern Chile. It is generally assumed that arsenic

has mainly a magmatic genesis trough volcanic activity and

ore deposits with high arsenic. Endogenous and exogenous

agents, such as meteorological effects on the soil, and the

circulation of subterranean water and wind, distribute

arsenic through the atmosphere, soil and water. Addition-

ally, the Atacama region is one of the largest mining areas

in the world and this type of activity also releases arsenic

(Flynn et al. 2002; Romero et al. 2003). Such natural and

industrial processes shape the global geochemical cycling

of arsenic in the region.

Communicated by A. Oren.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00792-012-0452-1) contains supplementarymaterial, which is available to authorized users.

J. Lara � M. Ferrero

Planta Piloto de Procesos Industriales Microbiologicos

(PROIMI), Consejo Nacional de Investigaciones Cientıficas y

Tecnicas (CONICET), Tucuman, Argentina

L. Escudero Gonzalez � C. Demergasso (&)

Centro de Biotecnologıa ‘‘Profesor Alberto Ruiz’’,

Universidad Catolica del Norte, Avda. Angamos 0610,

Antofagasta, Chile

e-mail: [email protected]

L. Escudero Gonzalez � C. Demergasso

Centro de Investigacion Cientıfica y Tecnologica para Minerıa

(CICITEM), Antofagasta, Chile

G. Chong Dıaz

Departamento de Ciencias Geologicas, Universidad Catolica del

Norte, Antofagasta, Chile

C. Pedros-Alio

Departament de Biologia Marina i Oceanografia, Institut de

Ciencies del Mar, CSIC, ES-08003 Barcelona, Spain

123

Extremophiles

DOI 10.1007/s00792-012-0452-1

Author's personal copy

http://dx.doi.org/10.1007/s00792-012-0452-1

Arsenic is a highly toxic element that supports a surprising

range of biogeochemical transformations. The biochemical

basis of these microbial interactions lies in energy yielding

redox biotransformations that cycle between the As(V) and

As(III) oxidation states. Those biotransformations have a

subsequent impact on the chemistry and the microbiology of

studied environments (Lloyd and Oremland 2006). Microbes

primarily metabolize inorganic arsenic either for resistance

or for energy generation. The transformations may involve

oxidation, reduction, methylation or demethylation (Slyemi

et al. 2011).

The microbial communities involved in arsenic metab-

olism have been extensively studied in saline alkaline (pH

8.5–9.8) environments in the US (Oremland et al. 2004;

Kulp et al. 2008), but poorly investigated in saline envi-

ronments at lower pH (7.8–8.6), like Salar de Ascotan,

Chile (pH 7.8–8.6) (Chong et al. 2000; Demergasso et al.

2007). It is worth considering that no alkaline brines are

known in Chilean salt flats because of the higher sulfate

and the lower alkalinity of inflow waters, as a consequence

of the suspected higher sulfur content in Chilean volcanic

rocks (Risacher and Fritz 2009).

Local cycling of arsenic was described in saline soda

lakes located in the Mojave Desert: Mono Lake and Searles

Lake (Oremland et al. 2004, 2005). Those transformations

included heterotrophic and lithotrophic arsenate respiring

microorganisms, as well as arsenic oxidizing microorgan-

isms. Recently, anoxygenic photosynthetic microorganisms

fueled by As have been isolated (Kulp et al. 2008). Organic

matter, sulfide, hydrogen, and As(III) (Hoeft et al. 2004)

were postulated as the main electron donors in such envi-

ronments, while As(V), Se (VI), sulfate, low O2 and nitrate

were the electron acceptors (Oremland et al. 2004, 2005). It

was shown that borate also plays an important role in the

arsenic cycle because of its apparent effect on sulfate

reduction (Kulp et al. 2007). The formation of arsenic III/

sulfide soluble species also drives the arsenic cycle in soda

lakes (Oremland et al. 2004). Considering the impact of the

pH on the borate and arsenic III/sulfide species solubility, a

different structure of the arsenic cycle should be expected

at lower pH.

In the brines of the Salar de Ascotan arsenic concen-

trations reach levels up to 2.5 mM. The occurrence of

arsenic microbial reduction associated to the formation of

arsenic sulfides (Demergasso et al. 2007), as well as the

arsenite-oxidizing microbial activity (Blamey, J. Biosci-

ence F., personal communication) have been already

described in this environment.

The aim of this study was to investigate the key

microorganisms associated with the arsenic cycle present

in the sediments of the Salar de Ascotan and Salar de

Atacama and the conditions to grow those populations. A

cultivation-based approach using organic electron donors

was used in that stage of the research. The composition of

the arsenic resistant and metabolizing bacterial populations

in aerobic and anaerobic enrichment cultures was analyzed

by 16S rDNA PCR-DGGE.

Materials and methods

Site description, sampling and measurements

A wide range of hypersaline environments with different

characteristics can be found in the Atacama Desert area,

Northern Chile. From west to east, deposits from the

Coastal Range, the Central Depression, the Pre-Andean

Depression, and the Altiplano Salt Deposits are found in

succession (Chong 1984). All these basins receive their

main water input from the east, which has a high per-

centage of salts from the leaching of volcanic rocks. The

combination of both arid climate and closed basins in these

salt flats produces a high rate of evaporation and the con-

sequent increase in salt content of the brines.

The evaporitic basins of the Salar de Ascotan (21�290S/

68�190W, 3.720 masl) and the Salar de Atacama (23�230S/

68�210W, 2.305 masl) have been described as Andean and

Pre-Andean salt flats, respectively (Stoertz and Ericksen

1974; Chong 1984). Ascotan (234 km2) is in the lowest

part of a tectonic basin surrounded to the east and west by

volcanic chains, including some active volcanoes over

5000 m high, with the highest peaks of about 6000 m. The

geological setting is dominated by volcanic structures that

include acidic (rhyolites) and intermediate (andesites)

rocks of Tertiary and Quaternary age. It is characterized by

its concentric mineralogy, with a very irregular distribution

of shallow ‘‘evaporites’’ (Herrera et al. 1997). The salt flat

exhibits both permanent and temporary shallow ponds, and

salt crusts, with a wide range of salinities and chemical

compositions.

The Salar de Atacama Depression is the oldest and the

largest evaporitic basin in Chile. It is located in a tectonic

intramontane basin filled with Tertiary/Quaternary clastic

and evaporitic sediments of continental origin. Its geolog-

ical setting is quite complex and receives both surface and

underground water inputs, mainly from the east. The main

input is related to the leaching and direct contribution of

acidic to basic volcanic material, mainly Tertiary and

Quaternary. However, there is also an important input from

a geological setting of different lithology as well as varied

age.

These salt flats were visited in June 2006 during an

intensive sampling expedition. Water and sediment sam-

ples were collected to perform physicochemical analysis

and culture enrichments of arsenic-resistant and arsenic-

metabolizing bacteria. The four different sites sampled in

Extremophiles

123


Salar de Ascotan were two ponds (Laguna Turquesa and

point J1) and two springs (V6 and V10) (Fig. 1). Mean-

while, two ponds were sampled in the Salar de Atacama:

Laguna de Tebenquiche and Laguna Burro Muerto

(Risacher and Alonso 1996) (Fig. 1). Two sites were

selected for the enrichments, one from each system, con-

sidering the As content.

An Orion model 290 pH metre was used to measure

temperature and pH in situ. Salinity, conductivity, and total

dissolved solids (TDS) were determined using an Orion

model 115 conductivity meter. Dissolved oxygen was

measured with a Thermo Orion model 9708. Water sam-

ples and surface sediment (0–3 cm) were transferred to

plastic bottles and kept in an icebox with ice until pro-

cessing. Water samples for chemical analysis were filtered

through 0.45 lm pore-size 25 mm diameter filter (Milli-

pore, Billerica, MA, USA). Approximately 1 L of water for

each data point was filtered through several nitrocellulose

0.22 lm pore size, 47 mm diameter filters for DNA and

RNA extraction. Additional samples were fixed (3.7 %

formaldehyde) and filtered through polycarbonate 0.22 lm

pore size, 47 mm diameter filter (Millipore, Billerica, MA,

USA) for bacterial total counts. Sediment samples for DNA

and RNA extractions and for 40, 6-diamidino-2-phenylin-

dole (DAPI)-cell counting were treated as follows: sedi-

ment (10 g) was homogenized in a 150-mL Erlenmeyer

flask with 50 mL of 0.9 % NaCl and 60 lL of 100 %

Tween 20 for 30 min in a rotatory shaker (150 rpm) at

room temperature. Subsequently, the suspension was

transferred to a 50 mL-centrifuge tube and centrifuged at

50009g for 5 min. Supernatant was filtered through sev-

eral nitrocellulose 0.22 lm pore size, 47 mm diameter

filters (Millipore, Billerica, MA, USA). Filters for DNA

extraction and for bacteria counting with DAPI-stain were

kept at -80 �C. Total arsenic was determined by hydride

generation atomic absorption spectroscopy (HGAAS), and

the other cation concentrations using an atomic absorption

spectrophotometer (AAnalyst 800; Perkin-Elmer, Norwalk,

CT, USA). Sulfate and chloride contents were measured

by gravimetric and Mohr method (APHA et al. 1998),

respectively.

Counting of in situ bacteria and culturable

arsenic-reducing bacteria

Bacteria were counted by epifluorescence using DNA-

specific dye DAPI with a Leica DMSL microscope. Cul-

turable arsenic-reducing bacteria (AsRB) were detected by

most-probable-number (MPN) incubations using fresh

minimal medium (Newman et al. 1997) modified by

addition of 0.008 % yeast extract and amended after

autoclaving with sterile 20 mM sodium lactate, 10 mM

sodium sulfate (Na2SO4), 1 mM L-Cistein (C3H7NO2S)

and 1 mM dibasic sodium arsenate (Na2HAsO4.7H2O)

Fig. 1 Map of Salar de Ascotan (left), showing the location of Laguna Turquesa, J1, V6 and V10, and Salar de Atacama (middle) showing the

location of Laguna Burro Muerto and Tebenquiche. Inset the salt flats sampled are shown in a map of Northern Chile (right)

Extremophiles

123


under N2:CO2:H2 atmosphere (80:15:5, v/v) (Demergasso

et al. 2007). To obtain a similar salinity to that of Laguna

Turquesa, NaCl was added to obtain a value of TDS close

to 30 g L-1. The highest decimal dilution was 10-6 and

five tubes were analyzed for each data point. The tubes

were incubated in the dark at 28 �C. An abiotic control was

carried out in sterile medium without inoculum. The

presence of yellow precipitate was considered as a positive

result. These cultures took between 3 and 4 weeks to pre-

cipitate arsenic sulfides as a result of the reduction of

arsenate and sulfate.

Enrichment cultures

The sediment samples from Laguna Turquesa (Salar de

Ascotan) were chosen for enrichments because they

showed the highest concentration of total arsenic

(28 mg L-1). Burro Muerto sediment was used for the

enrichments of Salar de Atacama samples. Sediment (4 g)

was dispensed into 40-mL bottles filled to the top with

50 % diluted LB medium with appropriate concentration of

NaCl (according to the salinity of the sampling point) for

anaerobic enrichments. The average content of organic

matter measured in Salar de Ascotan sediment samples in

different sampling expeditions was 1.25 %, similar to the

final concentration in the diluted LB medium used in the

enrichments. For aerobic cultures, 125 mL bottles were

used with 30 mL of culture medium. The bottles were

then amended either with Na2AsO2 (0.5 or 10 mM) or

Na2HAsO4.7H2O (13 or 100 mM). Henceforth, the terms

As(III) and As(V) will be used in the text, the tables and

the figures to refer to the two arsenic species, respectively.

For anaerobic cultures, the bottles were capped and bub-

bled with N2 gas across the butyl cap with a syringe for a

few minutes to displace the oxygen and then they were

incubated in the dark, without agitation. The incubation

was done in a rotary shaker at 180 rpm for aerobic cultures.

Both cultures, anaerobic and aerobic, were maintained at 4

or 28 �C. Samples (1 mL) were withdrawn after 10 days

and after 30 days of incubation and they were transferred

to microcentrifuge tubes for subsequent DNA extraction.

The enrichment cultures in which sodium arsenate was

present showed some yellow precipitate after 15 days of

incubation, and precipitates became abundant after 30 days

of incubation, showing that reduction of arsenate had taken

place.

DNA extraction and PCR amplifications

High molecular mass DNA of high purity was extracted

from sediment samples. Sediments were previously treated

to separate cells from sediment particles. Sediment (10 g)

was dispensed into 100 mL bottles with 50 mL of NaCl

(0.9 %) and 60 lL of Tween 20 (100 %). The bottles were

incubated at room temperature for 30 min at 180 rpm.

Then, sediment was allowed to settle and the supernatant

was filtered through a nitrocellulose 0.22 lm pore size,

47 mm diameter filter (Whatman�). DNA was extracted

from filters using High Pure PCR Template Preparation Kit

(Roche). DNA extraction from cultures was performed by

the CTAB method (Ellis et al. 1999) using 2 mL of each

enrichment culture. The quality and quantity of DNA from

the several sources were checked in a 0.8 % agarose gel

after staining with ethidium bromide. DGGE oligonucleo-

tide primers 341F-GC (Escherichia coli 16S rDNA posi-

tions 341–357) and 518R (E. coli 16S rDNA positions

518–534) (Muyzer et al. 1993) were used to amplified the

V3 region of eubacterial 16S rDNA from whole enrichment

cultures and those from sediment samples. The PCR cycle

followed previously described conditions (Becker et al.

2006) and the enzyme GoTaqTM

DNA Polymerase (Pro-

mega Co., Madison, WI, USA) was used.

DGGE analysis

DGGE was performed using a D-Code system (Bio-Rad

Laboratories, Inc., Hercules, CA, USA) (Ferrero et al.

2010). About 20 lL (approximately 800 ng) of PCR-

product was loaded for most of the samples and the gels

were run at a constant voltage of 120 V at 60 �C for 4.5 h.

The gel was stained with SYBR� Gold (Molecular Probes,

Eugene, OR) and visualized with a Bio-Rad UV transillu-

minator. Digital images of the gels were captured with the

Quantity One software (version 4.3.1; Bio-Rad Laborato-

ries, Inc., Hercules, CA) for use in comparative image

analysis. The gel image analyses were performed with

Cross Checker (version 2.91; Wageningen UR Plant

Breeding, The Netherlands) to analyze the bacterial com-

munity structure.

The Range-weighted richness (Rr) values were calcu-

lated based on the total number of bands (N) and the

denaturing gradient between the first and the last band of

each pattern (Dg) (Marzorati et al. 2008). The values

obtained were compared pairwise between conditions and

systems, and hypothesis tests were run to identify signifi-

cant differences. The band pattern distributions of the

samples were used to construct a binary matrix. Cluster

analyses (WPGMA), based on percent similarity between

the samples, were conducted using the SPSS.

The bands selected for identification were carefully

excised from the gel with a razor blade under UV illumi-

nation. Then, they were placed in 30 lL TE buffer and they

were incubated for 15 min at -70 �C or overnight at

-20 �C to elute the DNA from the gel; 0.5–2.0 lL of eluate

was used as a template for PCR amplification with the

original primer set, 341F and 518R (without a GC clamp).

Extremophiles

123


Some PCR products obtained from the excised bands were

randomly re-run in DGGE gel to confirm their relative

position to the bands from which they were excised.

Sequencing was performed directly on PCR products

with the 341F primer in Macrogen (Macrogen Inc., Korea).

Phylogenetic analysis

The identity and similarity to the nearest neighbor of DGGE

band sequences were obtained using the BLAST (Basic

Local Alignment Search Tool) algorithm (Altschul et al.

1997) at the National Center for Biotechnology Information

(NCBI, http://www.ncbi.nlm.nih.gov/BLAST/). Phylotypes

were selected at four different levels of minimal sequence

identity: 98, 95, 92 and 89 % using cd-hit (Li and Godzik

2006). The sequences were aligned using the alignment tool

in Greengenes (http://www.greengenes.lbl.gov).

Partial sequences were inserted into the optimized and

validated tree available in ARB (derived from complete

sequence data) using the maximum-parsimony criterion

and a special ARB parsimony tool that did not affect the

initial tree topology. The respective ARB tools were used

to perform maximum parsimony (MP), neighbor-joining

(NJ) and maximum likelihood (ML) analyses for full

sequences. The results from the three types of analyses

were essentially identical and only the maximum likeli-

hood trees are shown. Bootstrap resampling analysis for

500 replicates was carried out to estimate the confidence of

tree topologies.

The relative abundance of the major groups (Gamma-

proteobacteria and Firmicutes) was calculated adding the

percentage of intensity of the DGGE bands with sequences

affiliated to those groups after the phylogenetic analysis.

Pairwise comparisons between samples were conducted

using paired t tests.

Sequence accession number

The nucleotide sequences identified in this study were depos-

ited in the EMBL nucleotide sequence database (GenBank/

EMBL/DDBJ) under the accession numbers from FR751005

to FR751037, from FR796494 to FR796542 and from

FR839347 to FR839372 for DGGE bands.

Results

The geographical location and the results of the physico-

chemical and microbial analysis of the brines and the

sediments from the studied salt lakes are summarized in

Table 1. The differences in temperature between the pond

and the spring samples from the Ascotan system were due

to thermalism (Risacher et al. 1999). The pH of the Ta

ble

1G

eog

rap

hic

allo

cati

on

,p

hy

sico

chem

ical

and

bio

log

ical

par

amet

ers

fro

mS

alar

de

Asc

ota

nan

dS

alar

de

Ata

cam

a

Sam

ple

Co

ord

inat

esD

ate

Alt

itu

de

UV

-BW

/m2

(28

0–

32

0n

m)

O2

(mg

L-

1)

pH

Tem

p.

(�C

)

Co

nd

uct

ivit

y

(mS

cm-

1)

TD

S

(mg

L-

1)

As

(mg

L-

1)

SO

42-

(mg

L-

1)

Cl-

(mg

L-

1)

MP

N

cou

nt

(Cel

lsg

-1)

DA

PI

cou

nt

(Cel

lsm

L-

1

Cel

lsg

-1)

J-1

Po

nd

57

79

21

E/

76

09

32

8N

22

/06

/

20

06

37

44

1.3

8.9

8.3

03

.90

17

50

3.4

68

34

56

17

90

3.9

3E

?0

6

6.0

3E

?0

7

Sp

rin

g1

05

77

78

2E

/

76

10

57

3N

22

/06

/

20

06

37

40

1.6

8.8

81

82

.85

13

90

1.6

36

61

84

92

30

1.5

0E

?0

6

1.9

2E

?0

8

Sp

rin

g6

57

71

80

E/

76

22

87

0N

22

/06

/

20

06

37

38

11

0.8

ND

16

1.0

44

49

30

.91

81

11

39

23

01

.20

E?

06

5.4

7E

?0

7

Lag

un

a

Tu

rqu

esa

57

21

71

E/

76

10

65

3N

22

/06

/

20

06

37

41

1.2

7.5

8.3

31

8.5

10

20

02

83

65

03

00

94

90

4.2

5E

?0

7

5.1

2E

?0

7

Lag

un

aB

urr

o

Mu

erto

50

44

35

3E

/

74

24

61

7N

01

/05

/

20

06

23

50

0.1

55

47

.89

.68

3.3

55

80

09

.62

16

50

02

80

00

ND

1.4

0E

?0

6

Lag

un

a

Teb

enq

uic

he

57

73

38

E/

74

41

80

4

01

/05

/

20

06

23

50

0.1

03

2.9

67

.08

24

.21

77

.11

13

00

01

.11

16

10

04

20

00

ND

6.4

0E

?0

6

ND

no

td

eter

min

ed

Extremophiles

123


http://www.ncbi.nlm.nih.gov/BLAST/

http://www.greengenes.lbl.gov

Ascotan brines associated with the sediments ranged

between 8.0 and 8.6 while the pH of the Atacama brines

was lower (B7.8). The Ascotan brines have been reported

to evolve following different evaporation pathways.

Laguna Turquesa is located in the border between two

zones described as following the sulfate neutral and the

sulfate alkaline evolution pathways (Risacher et al. 1999).

Meanwhile, the water evolution in Salar de Atacama was

reported to follow that of neutral brines (pH lower than 7.8)

(Risacher and Alonso 1996). The arsenic concentration in

Laguna Turquesa was the highest detected in this study.

Arsenic reduction activity

Arsenic sulfide precipitation (yellow precipitate) was

observed in all of the anaerobic enrichments inoculated

with both Laguna Turquesa and Burro Muerto sediments

while it was not observed in the uninoculated controls or in

the aerobic enrichments. This demonstrates the occurrence

of arsenic reduction microbial activity in the sediments

analyzed (Kuai et al. 2001).

DGGE pattern analysis

The DGGE analysis of 16S rRNA gene fragments from the

enrichments of sediment samples from the Ascotan

(Fig. 2a) and Atacama (gel not shown) salt lakes showed

the development of different bacterial communities from

each environment at the different culture conditions.

Between 3 and 25 bands per sample were found in each

enrichment culture.

The cluster analysis (UPGMA) of DGGE bands showed

that the enrichments carried out at the same temperature

clustered together (for example, Fig. 2b shows the den-

drogram analysis with the relatedness of DGGE bands

obtained from the Ascotan and Atacama sediments in

anaerobic enrichments). A second level of clustering was

observed between the enrichments supplemented with the

same arsenic species (Fig. 2b).

The ranged-weighted richness (Rr), useful to reflect the

carrying capacity of the system (Marzorati et al. 2008), was

calculated to compare DGGE analyses from different gels.

The Rr observed in the original sample from the Ascotan

system and from the enrichments ranged between 2 and 35

(Fig. 3), which corresponds to environments whose rich-

ness ranges from a particularly restricted to medium range-

weighted richness environments (Marzorati et al. 2008).

The Rr parameter range in Atacama (0.18–155.5),

observed in the original sample and in the enrichments

(Fig. 3), corresponded to environments in which richness

ranged from particularly restricted to high range-weighted

richness (Marzorati et al. 2008). Pairwise comparisons

between samples using paired t tests showed significant

higher (\0.05) Rr values in the Atacama than in the

Ascotan samples.

In the anaerobic experiments inoculated with the

Ascotan sediments and supplemented with As(V) at 28 �C,

the Rr value was higher than 30 during the 30 days of

experiment. This is the limit established between a medium

range-weighted and a typical or very habitable richness

environment (Fig. 3).

Phylogenetic diversity

The identities of members of the bacterial communities

selected by the different enrichment conditions were

obtained by excision and sequencing of both representative

(e.g., most frequent) and rare bands from the DGGE gels.

Approximately 70 % of DGGE band sequences recovered

were similar to ‘‘uncultured bacteria’’ sequences. About 90

and 66 % of the OTUs found in each system (at 98 and

95 % sequence identity, respectively) were only detected in

that system.

According to phylogenetic analysis, the bacterial com-

munity in the original sediment sample from Laguna Tur-

quesa was composed of unidentified microorganisms,

Bacteroidetes, Proteobacteria and Firmicutes. Meanwhile,

the predominant microbial populations found in the sedi-

ments from Laguna Burro Muerto were related to Proteo-

bacteria, unidentified microorganisms, High GC Gram

positive and Firmicutes.

Firmicutes

The Firmicutes populations from Salar de Ascotan were

significantly (p \ 0.001) richer in anaerobiosis compared to

aerobiosis, considering similar physicochemical conditions

(Table 2). It was also richer at 4 �C than at 28 �C (p \ 0.05)

(Table 2). Firmicutes grew in all the conditions assayed

(arsenic species and concentrations, temperature and incu-

bation times) during anaerobic incubations. The Firmicutes

population in the Salar de Atacama samples was also sig-

nificantly (p \ 0.001) enriched in anaerobiosis compared to

aerobiosis (Table 2), considering similar physicochemical

conditions. Firmicutes grew better in 28 �C enrichments in

all the conditions assayed (arsenic species and concentra-

tions, and incubation times) during anaerobic incubations

compared to enrichments at 4 �C (p \ 0.05) (Table 2). In

addition, the Firmicutes populations in the cultures inocu-

lated with the Salar de Atacama samples decreased signifi-

cantly (p \ 0.05) at higher arsenic concentration. This

feature was not observed in the cultures inoculated with the

Ascotan samples.

According to DGGE band sequencing, four bacterial

sequences (102, 107, 108 and 86) recovered from

enrichment cultures (Tables 2 and S1) were closely

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123


related to cultured species from the Alkaliphilus genus

(Fig. 4a), whose type species is Alkaliphilus transvaal-

ensis (Takai et al. 2001). The Alkaliphilus-related

sequences recovered from the DGGE gel showed

between 96 and 97 % similarities to the Alkaliphilus

transvaalensis and Alkaliphilus metalliredigens QYMF

and they were relatives of Alkaliphilus oremlandii

(Clostridium sp. OhILAs), a microorganism able to

metabolize arsenic (Fisher et al. 2008).

The closest cultured relative of two other sequences

(14 and 90) was Sporacetigenium sp (Tables 2 and S1,

Fig. 4a). These sequences were also related to Pepto-

streptococcus sp. Ch5, isolated from the Andean salt lakes

(Fig. 4a). The Clostridiaceae bacterium CAa338 clustered

with six sequences (106, 80, 262, 224, 227 and 223)

retrieved from the experiments (Tables 2 and S1). The

strain CAa338 is an alkaliphilic bacterium isolated from

tufa columns from Greenland (Schmidt et al. 2006). Four

b

a

Fig. 2 a Negative image of a denaturing gradient gel electrophoresis

(DGGE) of bacterial community in cultures supplemented with

As(III) or As(V) inoculated with the sediment samples from Laguna

Turquesa. Enrichment conditions are indicated at each lane. Bands

that were cut off from the gel are labeled with the same number as in

Table S1. When bands across several lanes could be identified as

being the same, they all have the same number. Turq and J1-Sed

correspond to original sediment samples from Laguna Turquesa and

J1 from Salar de Ascotan, respectively. b Relationship between

bacterial community structures represented by a dendrogram (UP-

GMA cluster analysis) obtained from anaerobic enrichments inocu-

lated with the Ascotan (left) and the Atacama (right) sediments

Extremophiles

123


other sequences (64, 65 70 and 283) clustered together with

a psychrophilic anaerobic bacterium isolated from rice

paddy soil (Clostridium sp. C5S18). One of the closest

cultured relatives of such clusters is a representative of

Clostridiaceae family, Caloramator sp. (Fig. 4a). Three

sequences (37, 39 and 61) formed a cluster (Bacillus 1)

with Bacillus agaradhaerens (Table S1, Fig. 4a). B. aga-

radhaerens is a starch-hydrolyzing alkaliphilic bacterium

isolated from soda lakes (Martins et al. 2001). This bac-

terium was described as an halotolerant, aerobic, hetero-

trophic and alkaliphilic dissimilatory Fe(III)-reducing

bacterium, which was isolated from the Great Salt Plains of

Oklahoma (Caton et al. 2004).

In the aerobic enrichments inoculated with the Ascotan

samples, B. agaradhaerens-related sequences (37 and 39)

were the most abundant from the Firmicutes phylum in

almost all the conditions tested.

Another cluster (Bacillus 2) is formed by sequences (13,

17, 18 and 29) isolated from the enrichments (Table 2) and

a microorganism that has been previously found in saline

lakes (Bacillus sp. N29) (Ordonez et al. 2009). The closest

cultured relatives were the strict aerobes B. horikoshii

(Nielsen et al. 1995), B. arsenicus (Shivaji et al. 2005) and

the facultative anaerobe B. selenatarsenatis (Fig. 4a). The

abundance of these sequences was higher at 4 �C.

B. arseniciselenatis (Switzer Blum et al. 1998) isolated

from Mono Lake formed a cluster (Fig. 4a) with five more

sequences from the enrichments (69, 73, 77, 97 and 252).

Most of these (69, 73 and 77) were retrieved only in the

experiments at 4 �C. Only one sequence (band 74) related to

Paraliobacillus (similarity of 96 %) was found in the

enrichments. Another phylotype (277 and 163) that was

related to Halolactibacillus miurensis (Table S1, Fig. 4a) was

obtained from enrichments and from the original sediment.

Halolactibacillus and Paraliobacillus genera are included in

a phylogenetic group composed of halophilic/halotolerant/

alkaliphilic and/or alkalitolerant species in Bacillus rRNA

group 1. The genus Paraliobacillus was first proposed

(Ishikawa et al. 2002) to accommodate a Gram positive,

facultatively anaerobic, chemo-organotrophic, spore-form-

ing and slightly halophilic bacterium (Chen et al. 2009).

Another sequence (271) was distantly related to micro-

organism from the arsenate-reducing sulfide-oxidizing

population of Mono Lake (Hollibaugh et al. 2006). This

sequence was not included in the phylogenetic tree in

Fig. 4a for clarity.

Proteobacteria

Gammaproteobacteria-related sequences were predominant

in most of the aerobic enrichments inoculated with the

Ascotan sediments (p \ 0.01) (Table 2). In the anaerobic

cultures, the sequences closely related to Gammaproteo-

bacteria were less abundant than the sequences related to

other groups (mainly Firmicutes) (Table 2).

There were also significant differences between the

Gammaproteobacteria populations in aerobic and anaero-

bic conditions in the experiments inoculated with the

Atacama samples. Gammaproteobacteria populations from

Salar de Atacama were significantly (p \ 0.05) enriched in

aerobic compared to anaerobic environments (Table 2).

The uncultured marine bacteria (clone A3 and A50 from

GenBank) formed a cluster with sequences obtained from

the enrichments with the Ascotan (40, 41, 42, 59, 60 and

205) and the Atacama sediments (Fig. 4b). Several Gam-

maproteobacteria sequences from the cultures (6, 7, 8, 34,

35, 21, 12, 171 and 182) had Halomonas as their closest

relative (Table S1, Fig. 4b) and they also clustered with the

Fig. 3 Range-weighted

richness (Rr) of the denaturing

gradient gel electrophoresis

(DGGE) profiles of enrichments

inoculated with the Ascotan

(left) and the Atacama (right)sediments and amended with

organic matter and As in aerobic

and anaerobic conditions

Extremophiles

123


strain GFAJ-1 isolated from Mono Lake (Wolfe-Simon

et al. 2010). One Halomonas-related sequence was also

obtained from the original Ascotan sediment sample.

One more cluster was formed by eight sequences from

Atacama enrichments (127, 173,162,147,129,164,165 and

135) plus cultured and uncultured representatives of Mar-

inobacter genus (Table S1, Fig. 4b), some of them isolated

from hydrothermal vents (Takai et al. 2005).

The closest relatives of three other sequences of the

Gammaproteobacteria group retrieved from the cultures

(91, 105 and 274) were the uncultured Pseudomonas sp.

clone ESP450-K6IV-53, isolated from the deep oxycline

(450 m depth) of the eastern tropical South Pacific

(Stevens and Ulloa 2008).

A few Vibrio-related sequences (146 and 145), not

included in the phylogenetic tree for clarity, were also

isolated from the aerobic enrichments inoculated with the

Atacama sediments (Table S1).

The sequence related to Alphaproteobacteria group (22)

showed the highest phylogenetic similarity with a soil

microorganism that is able to fix nitrogen (Saikia et al.

2007). The nearest neighbors of the predominant Alpha-

proteobacteria sequences (215, 217, 219, 294, 292, 297 and

222) were related to Rhodobacteracea.

Table 2 Distribution of the retrieved sequences among the enrichment cultures inoculated with the Ascotan and the Atacama sediments

The number of ? indicates significant differences in the relative abundances of Firmicutes and Gammaproteobacteria sequences

The dark cells mean the presence of those sequences in the culture condition

Extremophiles

123


The closest relative of one sequence (100) (Table S1,

Fig. 4b) was an uncultured microorganism (clone RH.208-

35-22) isolated from hypersaline industrial water (Gen-

BanK information). The most closely related (93–98 %)

cultured microorganisms were Arcobacter marinus (Kim

et al. 2010). The Epsilonproteobacteria-related sequences

were more abundant in the aerobic enrichments inoculated

with the Atacama samples. Three other sequences (123,172

and 263) clustered together with that group (Fig. 4b). Five

sequences (195, 148,196, 170 and 150) formed another

cluster without cultured relatives (Fig. 4b). The closest

cultured organisms also belonged to the Arcobacter genus

(Donachie et al. 2005; Kim et al. 2010). Another sequence

from the Epsilonproteobacteria group (175, not included in

the phylogenetic analysis) was retrieved and its closest

relative (Table S1) was found in a metagenome of a

ubiquitous and abundant but uncultivated oxygen mini-

mum zone microbe (SUP05) (Walsh et al. 2009).

Two other sequences (24 and 41) belonged to the

Betaproteobacteria group (Table S1, Fig. 4b) and the

closest culture relative is Burkholderia sp., isolated from

Vietnamese soils (Huong et al. 2007). One of these

sequences was also retrieved from the original Ascotan

sample and it represented 30 % of the bacterial

community.

One Desulfovibrionaceae (Deltaproteobacteria) related

sequence (241) was also found and the closest cultured

microorganism was a sulfate reducing bacteria Desulf-

ovibrio sp. AR1201/00 (Table S1, Fig. 4b), isolated from

sediment samples (GenBank information).

Other groups

The Bacteroidetes (Table S1, DGGE bands 1, 47, 53, 55,

110, 112 and 113) group was present in point J1 and in

Laguna Turquesa sediment samples (Ascotan). However,

no sequences from this particular group were enriched in

cultures supplemented with arsenic, neither aerobic nor

anaerobic.

High GC-related sequences (218, 221, 115 and 118)

were retrieved from Tebenquiche sediment samples but not

from the Laguna Burro Muerto sediments. One sequence

(244) related to Synergistetes Phylum (Vartoukian et al.

2007) was found in the cultures. The closest cultured

(97 %) is Dethiosulfovibrio acidaminovorans strain sr15

(Table S1) isolated from sulfur mats in saline environments

(Surkov et al. 2001). That strain was reported as strictly

anaerobic, sulfur- and thiosulfate-reducing bacteria.

Another sequence (103) affiliated to Spirochaetes was

retrieved. The closest relative of this sequence (Table S1)

Fig. 4 Phylogenetic tree that includes partial sequences from DGGE bands and clones affiliated to the predominant bacterial phyla, Firmicutes

(a) and Proteobacteria (b). Bar corresponds to 0.1 substitutions per nucleotide position

Extremophiles

123


was isolated from deep sea sediments (Toffin et al. 2004) as

a dominant community member together with Firmicutes

and Proteobacteria sequences. The members of the Spiro-

chaetes were reported to inhabit biotopes with high salin-

ity, alkalinity, pressure, and temperature in methanogenic

reactors and in sediments under sulfate-reducing conditions

(Fernandez et al. 1999; Hoover et al. 2003; Toffin et al.

2004).

Sequences without relatives in the databases (2, 125,

185, 225, 226, 233, 242, 243, 276 and 278) were also

detected (Table S1). The occurrence of these sequences in

the culture experiments is summarized in Table 2.

Discussion

To understand the microbiome associated with the As cycle

in saline circumneutral environments, we compared the

behavior of the microbial communities from two similar

systems amended with As and organic matter. The enrich-

ment conditions had different effects on the Ascotan and the

Atacama communities.

Molecular fingerprinting techniques—such as DGGE—

are useful to study the structure and the composition of the

microbial communities. The need to interpret and compare

the information obtained from such type of techniques has

Fig. 4 continued

Extremophiles

123


led to the development of a three stage tool set successfully

used in several environments (Read et al. 2011). The tool

was designed for the DGGE pattern analysis (Marzorati

et al. 2008) and it allows analysis of microbial fingerprints

obtained with different gel runs. The interpretation is based

on three parameters: (1) the ranged-weighted richness (Rr),

useful to reflect the carrying capacity of the system

(Marzorati et al. 2008); (2) the dynamics (Dy) reflecting

the specific rate of species coming to significance and (3)

the functional organization (Fo), recently renamed as

community organization (Co) and defined as the ability of

the community to organize itself in an adequate distribution

of dominant microorganisms and resilient ones, condition

that should assure the potentiality of counteracting the

effect of a sudden stress exposure (Marzorati et al. 2008).

This provides an ecological interpretation of the raw

DGGE data describing the structure of the community

(Read et al. 2011). Thus, it was possible to derive con-

clusions about the relationships between the structure of

the microbial community and its functionality.

In spite of the higher values of the Rr parameter in the

Atacama microbial communities, the Rr parameter of

specialized populations in the Ascotan samples increased

under anaerobic conditions (Fig. 3) during the incubation

period (from 10 to 30 days). These results indicate a

stimulation of the native microbial community by organic

matter and As(V) in anaerobiosis.

The salinity level of both enrichment media seems to be

one of the causes of the differences between the cultures

inoculated with the Ascotan and the Atacama sediments.

Following Oren’s theory (Oren 1999), anaerobic processes,

such as sulfate reduction, yield insufficient energy to sup-

port the metabolism of these microorganisms under salt-

saturated conditions. In similar analyses performed in

sediments from two salt lakes in the US (Kulp et al. 2007),

the authors concluded that when salt concentration

approaches salt saturation levels, microbial metabolisms

based on bioenergetically favorable electron acceptors

(such as arsenate) may become more important (Kulp et al.

2007).

Firmicutes-related sequences from the Ascotan and the

Atacama environments showed differential traits in anaer-

obic enrichments.

The increase of the Rr parameter between 10 and 30 days

of incubation in anaerobic enrichments inoculated with the

Ascotan sediments was mainly due to the appearance of

new Firmicutes sequences. The changes in abundance of

major Firmicutes sequences suggest that psychrophilic,

and mesophilic or psychrotolerant organisms occur in the

Ascotan and the Atacama samples, respectively (Table 2).

On the other hand, the arsenic concentration introduces a

most important shift in the abundance of the major

sequences in the Atacama compared to the Ascotan

anaerobic enrichments (data not shown). Those features

could be due to the higher arsenic concentrations and the

lower temperatures usually registered in Ascotan.

The Gammaproteobacteria-related sequences seemed to

be responsible for most of the microbial growth with

arsenic occurrence in aerobic enrichments inoculated with

the Ascotan and the Atacama samples. The most abundant

sequences from Gammaproteobacteria phylum were rela-

ted to Marinobacter and Halomonas in the Atacama and

the Ascotan enrichments, respectively (Table 2). Marin-

obacter is restricted to the enrichments from the Atacama

sediments; meanwhile, Halomonas grew better in the

cultures inoculated with the sediments from Ascotan

(Table 2). Halomonas and Marinobacter have been

reported to be cosmopolitan genera commonly found in

the deep sea and in hydrothermal vent settings (Kaye

et al. 2010). The most abundant Gammaproteobacteria

sequences in Ascotan, the system with higher As levels in

the aerobic and the anaerobic enrichments, belonged to

Halomonas, Pseudomonas and an uncultured marine

bacteria. It is worth mentioning that reports about arsenic

resistance in Halomonas (Takeuchi et al. 2007), as well as

in Pseudomonas (Cai et al. 1998, 2009), have suggested

the presence of an unknown resistance mechanism. The

accumulation of As has been reported in members of

the Halomonadaceae family (Takeuchi et al. 2007). The

Halomonas-related sequences disappeared at 100 mM

As(V) in aerobic experiments with the Ascotan samples.

Only the sequences related with marine bacteria (clone A3

and A50 from GenBank) remained in those enrichments.

The analysis of the ecotype distribution of microorgan-

isms from the Marinobacter and Halomonas lineages in

hydrothermal settings suggested an antagonistic effect of

higher concentrations of hydrogen sulfide on Marinob-

acter (Kaye et al. 2010).

The results suggest that Halomonas-related microor-

ganisms were psychrophilic (significant—\0.01—higher

abundances were observed at 4 �C) and that Marinobacter

related microorganisms were psychrotolerant (Fig. 5). The

range of growth temperature reported for several micro-

organisms from Marinobacter genus is between 0 and

45 �C (Montes et al. 2008; Zhang et al. 2008). On the

other hand, Halomonas-related sequences increases their

abundance preferentially in experiments supplemented

with As III (data not shown). Meanwhile, Marinobacter

strains grew in both As(III) and As(V) supplemented

enrichments. In addition, Gammaproteobacteria closely

related to Halomonas have been reported to be abundant

in contaminated (de Souza et al. 2001; Dib et al. 2010)

and cold environments (Reddy et al. 2003). Compara-

tive studies suggest that most of the members of Halo-

monas are strictly aerobic organisms (Bouchotroch et al.

2001).

Extremophiles

123


Most of the sequences associated to known arsenic

respiring microorganisms were obtained from the enrich-

ments inoculated with the Ascotan sediments and belon-

ged to the Firmicutes phylum (Table 2). The related and

known arsenic respiring microorganisms from the Firmi-

cutes phylum are Alkaliphilus oremlandii, Caloramator

sp., Bacillus selenatarsenatis and Bacillus arseniciselena-

tis. Alkaliphilus oremlandii (Clostridium sp. OhILAs)

is able to use arsenate and thiosulfate as terminal elec-

tron acceptors, and it has the arrA gen, involved in

anaerobic-arsenate-reducing metabolism (Fisher et al.

2008) (Fig. 4a; Table 2). Caloramator sp. metabolizes

arsenic and produces beta-realgar (arsenic sulfide), a

mineral that has been recently observed as a by-product of

arsenic microbial metabolism (Ledbetter et al. 2007a;

Ledbetter et al. 2007b). The characteristics of this mineral

were similar to those of pararealgar, which was previously

identified as a by-product of microbial precipitation of

arsenic sulfides using bacterial cultures from Ascotan

(Demergasso et al. 2007). Some microorganisms from the

Caloramator genus have been also reported to be aceto-

genic (Drake et al. 2008). B. selenatarsenatis is a selenate

and arsenate-reducing bacterium (Yamamura et al. 2007).

B.arseniciselenatis grows by dissimilatory reduction of

As(V) to As(III) with the concomitant oxidation of lactate

to acetate plus CO2.

Only two sequences compatible with arsenate-reducing

microorganisms were isolated from the enrichments inoc-

ulated with the Atacama sediments (Table 2). In addition,

the reports of an arsenate-respiring and arsenite-oxidizing

marine species, Marinobacter santoriniensis sp. isolated

from hydrothermal sediments (Handley et al. 2009), allow

us to infer that Marinobacter-related sequences could be

also associated to arsenic-metabolizing microorganisms.

Other sequences inside the Firmicutes phylum are related

to cultured microorganisms with known metabolisms that

could play indirect roles in the arsenic biogeochemical cycle

like Sporacetigenium sp and Halolactibacillus (Table 2).

The isolated strains of the Sporacetigenium genus produced

H2, acetic acid and ethanol from glucose fermentation (Chen

et al. 2006). Acetogens had traditionally been considered to

be strict anaerobes. Recently, however, these microorgan-

isms have been also found in environments subject to fluc-

tuations in O2, such as aerated soils (Gossner et al. 2008).

Halolactibacillus miurensis is a halophilic and alkaliphilic

marine lactic acid bacterium (Ishikawa et al. 2005). Other

fermentative microorganisms from the Proteobacteria phy-

lum could be represented by the sequences clustered with

Arcobacter marinus (Kim et al. 2010). A. marinus grows well

under either aerobic or microaerobic conditions and forms

a group with the fermentative and facultative anaerobic

Epsilonproteobacterium, Arcobacter halophilus (Donachie

et al. 2005).

Those fermentation products could be potential sources

of electrons for arsenic reduction.

The Alphaproteobacteria-related sequences clustered

with Rhodobacteraceae that are able to decompose organic

compounds under visible light with simultaneous evolution

of hydrogen and carbon dioxide (Seifert et al. 2010).

In addition, sulfate-reducing and sulfur- and thiosulfate-

reducing bacteria like Desulfovibrio sp. (Deltaproteobac-

teria) and Dethiosulfovibrio acidaminovorans strain sr15

(Synergistetes) (Table 2) could also play indirect roles in

the arsenic biogeochemical cycle.

The phylogenetic analysis also revealed the occurrence of

sequences clustering with each other and without close cul-

tured relatives, mainly from Firmicutes and Proteobacteria

phyla in both the Ascotan and the Atacama enrichments

(Fig. 4).

The phylogenetic analysis of the sequences retrieved from

the Ascotan and the Atacama enrichment experiments

showed that the populations involved belonged to the Phyla

represented in the phylogenetic tree of the arsenotrophs

(Oremland et al. 2009) (Table 2; Fig. 4). In addition, Spi-

rochaetes and Synergistetes sequences were also retrieved and

they could be new targets of isolation efforts together with

Halomonas and Marinobacter in the Gammaproteobacteria

Fig. 5 Marinobacter and Halomonas relative abundance percentages

in the enrichments inoculated with the Atacama and Ascotan

sediments, respectively. Each box encloses 50 % of the data with

the median value of the variable displayed as a line. The top and

bottom of the box mark the limits of ±25 % of the variable

population. The lines extending from the top and bottom of each box

mark the minimum and maximum values within the data set that falls

within an acceptable range. Any value outside of this range, called an

outlier, is displayed as an individual point (KaleidaGraph tool)

Extremophiles

123


Phylum, which were found to be a predominant group in the

Ascotan and the Atacama communities, respectively. Isola-

tion of such microorganisms will allow the confirmation of

their hypothetical participation in the arsenic biogeochemical

cycle or its arsenic tolerance.

Conclusions

Fingerprint techniques were useful to preliminary assess

the microbial community structure and function in the

arsenic bearing salt lakes of circumneutral pH, and to

design the research strategy to understand the biogeo-

chemical cycle of that element.

Based on the stimulation of populations from the

Ascotan microbial community in the enrichment conditions

and on the phylogenetic relationship of some of those

populations with reported sequences, we can conclude that

a better established arsenic cycle should occur in Ascotan.

Microbial populations compatible with microorganisms

able to transform arsenic to conserve energy, accumulate

arsenic, produce H2, H2S and acetic acid—potential sour-

ces of electrons for arsenic reduction—, and to tolerate

high arsenic levels is evidence for a fully developed As

cycle.

The predominant ecotypes that were specifically asso-

ciated with transformation of organic carbon in those

arsenic rich salt lakes in Northern Chile belonged to the

Firmicutes and Proteobacteria phyla. Further culturing

efforts should be directed toward the isolation of repre-

sentatives of those predominant. In accordance with the

results obtained, salinity, temperature, and oxygen avail-

ability are some of the selective conditions that should be

used to isolate the target populations. Similar enrichment

experiments are needed to investigate the autotrophic and

phototrophic populations involved in the arsenic cycle in

these systems.

Acknowledgments This work was supported by funds provided by

the BBVA Foundation project BIOARSENICO and the Chilean

Government through FONDECYT Project 1100795 from the Science

and Technology Chilean Commission (CONICYT). We also would

like to thank the A. v. Humboldt Stiftung for the donation of part

of the equipment used in this project, as well as V. Iturriaga,

M. J. Demergasso and M.V. Coalova for their collaboration.

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